Process for Manufacturing a Sweetener and Use Thereof

ABSTRACT

Highly purified Stevioside, Rebaudioside A and a purified sweet steviol glycoside mixture were prepared from sweet glycoside extracts obtained from  Stevia rebaudiana  Bertoni leaves. The resulting sweeteners are suitable as non-calorie, non-cariogenic, non-bitter, non-lingering sweeteners, which may be advantageously applied in foods, beverages, and milk products.

RELATED APPLICATIONS

This application is a continuation-in-part application of and claims thebenefit of priority to U.S. patent application Ser. No. 11/246,066,filed Oct. 11, 2005; U.S. patent application Ser. No. 11/246,152, filedOct. 11, 2005; U.S. provisional application Ser. No. 61/260,593, filedNov. 12, 2009; U.S. provisional application Ser. No. 61/290,778, filedDec. 29, 2009; U.S. patent application Ser. No. 12/684,129, filed Jan.8, 2010; U.S. patent application Ser. No. 12/684,130, filed Jan. 8,2010; and U.S. patent application Ser. No. 12/684,981, filed Jan. 11,2010.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for producing a highlypurified sweet steviol glycoside mixture, Stevioside and Rebaudioside Afrom the extract of the Stevia rebaudiana Bertoni plant and use thereofin various food products and beverages.

2. Description of the Related Art

In view of food sanitation, the use of artificial sweeteners such asdulcin, sodium cyclamate and saccharin has been restricted. Howevernatural sweeteners have been receiving increasing demand. Steviarebaudiana Bertoni is a plant that produces an alternative sweetenerthat has an added advantage of being a natural plant product. Inaddition, the sweet steviol glycosides have functional and sensoryproperties superior to those of many high potency sweeteners.

The extract of Stevia rebaudiana plant contains a mixture of differentsweet diterpene glycosides, which have a single base-steviol and differby the presence of carbohydrate residues at positions C13 and C19. Theseglycosides accumulate in Stevia leaves and compose approximately 10%-20%of the total dry weight. Typically, on a dry weight basis, the fourmajor glycosides found in the leaves of Stevia are Dulcoside A (0.3%),Rebaudioside C (0.6%), Rebaudioside A (3.8%) and Stevioside (9.1%).Other glycosides identified in Stevia extract include Rebaudioside B, C,D, E, and F, Steviolbioside and Rubusoside.

The physical and sensory properties are well studied only for Steviosideand Rebaudioside A. They were tested for stability in carbonatedbeverages and found to be both heat and pH stable (Chang and Cook,1983). The sweetness potency of Stevioside is around 210 times higherthan sucrose, Rebaudioside A in between 200 and 400 times, andRebaudioside C and Dulcoside A around 30 times (Phillips, 1989 andTanaka, 1997).

However, apart from its high level of sweetness, they have alsointrinsic properties of post-bitter taste and unpleasant and undesirableaftertaste. Some undesirable taste characteristics of glycosides can beas a result of contamination of other substances, presented in extract.

One of the main ways to improve the taste quality is the enzymaticglycosylation of mixture of semi-purified steviol glycosides. Anotherway to produce highly purified individual glycosides with standardcharacteristics and minimal content of accompanying compounds.

The invention related to the purification of two mainglycosides—Stevioside and Rebaudioside A and use thereof.

A process for the recovery of diterpene glycosides, including Steviosidefrom the Stevia rebaudiana plant is described (U.S. Pat. No. 4,361,697).A variety of solvents, having different polarities, were used in asequential treatment that concluded with a high performance liquidchromatographic (HPLC) separation procedure.

A method for the recovery of Rebaudioside A from the leaves of Steviarebaudiana plants has been developed (U.S. Pat. No. 4,082,858). Again,final purification is achieved by liquid chromatography subsequent to aninitial extraction with water and an alkanol having from 1 to 3 carboncarbons, preferably methanol. It is also known that water may be used asthe initial solvent; their preferred solvent at this stage is a liquidhaloalkane having from 1 to 4 carbon atoms. The preferred second solventis an alkanol having from 1 to 3 carbon atoms, while the preferred thirdsolvent is an alkanol having from 1 to 4 carbon atoms and optionallyminor amounts of water.

Individual sweet glycosides can be obtained from the Stevia rebaudianaplant. A mixture of sweet glycosides extracted from the Steviarebaudiana plant is processed to remove impurities by using two types ofion-exchangers. After removing the mixed sweet glycosides from thesecond column with methanol, the solution is dried. Upon refluxing thedried solids in a methanol solution and then cooling the solution,Stevioside precipitates out. The filtrate is further concentrated andcooled to precipitate out Rebaudioside A. This Rebaudioside A can befurther purified as can the previously obtained Stevioside (U.S. Pat.No. 5,962,678). However, a large amount of toxic organic solvent, suchas methanol is used.

However, all the above-mentioned methods allow the production ofStevioside and Rebaudioside A not in highly purified grade, whichfurther possess a residual bitterness and aftertaste.

On the other hand, the unfavorable taste of the glycosides can be as aresult of contamination of impurities, presented in extract. Highlypurified Stevioside and Rebaudioside A possessing an improved tasteprofile and there is a need to provide an easy and commercially valuableprocess for the manufacturing the highly purified Stevioside andRebaudioside A, and use thereof in various beverages and food products.

SUMMARY OF INVENTION

An object of the present invention is to provide a commercially valuableprocess for producing a highly purified sweetener from the extract ofStevia rebaudiana Bertoni plant and use thereof in various food productsand beverages, which overcomes the disadvantages of the related art.

The invention, in part, pertains to the dried and powdered leaves beingsubjected to water extraction and the resulted extracts is purifiedusing treatment with a base such as calcium hydroxide and then ironchloride. The filtrate was deionized on, e.g., Amberlite FPC23H,Amberlite FPA51, and Amberlite FPA98Cl. The filtrate is concentratedunder vacuum and spray dried. The isolation and purification ofStevioside and Rebaudioside A were developed using alcoholicprecipitation and ultrafiltration. The highly purified Stevioside andRebaudioside A were obtained. Any type of existing Stevia extract withvarious ratios of sweet steviol glycosides are feasible.

The highly purified glycosides were applied in various foods andbeverages as sweetener.

The invention, in part, pertains to a purified sweet glycosides extractproduced from the Stevia rebaudiana plant, wherein the main sweetglycosides are Rebaudioside A and Stevioside, obtained by a processincluding drying Stevia rebaudiana leaves, treating the leaves toextract an aqueous liquid solution containing mixed sweet steviolglycosides, extracting the Stevia rebaudiana leaves, obtaining anextract, filtering the extract, obtaining a filtrate, treating thefiltrate with a base such as calcium hydroxide, treating the extractwith trivalent iron chloride, desalting, decolorizing, and evaporatingthe filtrate to dryness.

In the invention, purified Rebaudioside A and Stevioside can be obtainedby dissolving sweet steviol glycosides in methanol at ambienttemperatures to precipitate Stevioside, filtering the solution torecover a precipitate of Stevioside, purifying, recovering a high purityStevioside, concentrating the remaining solution and evaporating todryness, suspending the powder in ethanol, heating and then cooling thesolution to precipitate Rebaudioside A. Suspending the crystallineRebaudioside A obtained in ethanol-water solution at cool conditions(10-12° C.) prepares a high purity of Rebaudioside A. Stevioside orRebaudioside A has a purity of at least 98%. Applications are found invarious foods such as chocolate, ice cream, beverage, dairy products, asa sweetener in a tablet form.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the invention. The drawings illustrate embodiments ofthe invention and together with the description serve to explain theprinciples of the embodiments of the invention.

FIG. 1 shows a sensory evaluation of raw Stevia extract, Stevioside, andRebaudioside A;

FIG. 2 shows a sensory evaluation of Stevioside with a different gradeof purity; and

FIG. 3 shows a sensory evaluation of Rebaudioside A with a differentgrade of purity;

DETAILED DESCRIPTION

Advantages of the present invention will become more apparent from thedetailed description given hereinafter. However, it should be understoodthat the detailed description and specific examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom this detailed description.

The dried leaves of Stevia rebaudiana Bertoni were extracted by 10volumes of water. The proportion of extraction water preferably wasabout 5 liters to about 15 liters (pH 6.0 to 7.0) to one kilogram ofleaves. Greater volumes of solvent can be used, however, it was notpreferable from the practical standpoint. The duration of extraction maybe from 0.5 hours to 24 hours, with a period of from about 1 hours toabout 6 hours preferred.

The extraction temperature can be in the limits of 25-90° C., howeverthe temperatures between 45-75° C. are more preferable.

The plant material was separated from the solution by filtration, andthe pH of the filtrate was adjusted to about 10 with calcium hydroxideand heated between 40-60° C., preferably from 50° C. to 55° C., forabout 0.5-1.0 hours, cooled to ambient temperature with slow agitation,and neutralized by FeCl₃. After mixing for 10-15 minutes, theprecipitate was removed by filtration; the filtrate was passed throughthe Celite, deionized, and decolorized by Amberlite FPC23H, AmberliteFPA51, and Amberlite FPA98Cl by conventional manner. The solution wasconcentrated and spray dried.

The resulting sweet steviol glycoside mixture was a yellow powder andhad a content of 3.4% Dulcoside, 64.6% Stevioside, 6.7% Rebaudioside Cand 25.3% Rebaudioside A.

An HPLC analysis of the obtained product was carried out using anAgilent Technologies 1100 Series (USA) equipped with Zorbax-NH₂ columnusing acetonitrile-water gradient from 80:20, v/v (2 minutes) to 50:50,v/v during 70 minutes and UV detector at 210 nm.

The obtained powder was dissolved in methanol and maintained at atemperatures 20-50° C., preferably at 20-25° C., for 0.5-6.0 hours,preferably 0.5-1.0 hours with agitation. The proportion of extract andmethanol was between 1:2-1:7, w/v, preferably 1:5. During this time theprecipitate was formed, which was filtered and dried. According to theHPLC analysis, the powder contents were around 90-91% of Stevioside. Asecond treatment by methanol was not efficient to prepare high purityStevioside.

For the further purification, the powder was mixed with two volumes of90% of ethanol and at 10-12° C. and maintained for about 30 minutes withslow agitation. The precipitate was separated by filtration and driedunder vacuum. The Stevioside with about 98.5-99.4% purity was obtained.

The filtrates were combined and used for recovery of Rebaudioside A. Forthis purpose the remaining solution was evaporated to remove themethanol, the syrup obtained diluted with water and passed throughpolysulfone based ultrafiltration membranes (with a filteringdiscrimination of 2.5 kDa) (Liumar Technologies, Ottawa, Canada) withdiafiltration. The filtrate was concentrated and spray dried. Theobtained powder was mixed with 96.2% ethanol and maintained at 45-50° C.for about 30 minutes with agitation. The proportion of syrup and ethanolwas between 1:2-1:7, w/v, preferably 1:5. During this time theprecipitate was formed, which was filtered and dried. Rebaudioside Awith 88-90% purity was obtained. For the further purification the powderwas mixed with two volumes of 92% ethanol and maintained at 10-12° C.for about 60 minutes with slow agitation. The crystals were filtered anddried. Rebaudioside A with 98.9% purity was obtained.

Based on the results of preliminary test on the sweetening power of thesweeteners, aqueous solutions of commercial Stevia extract (0.05%)commercialized by Ganzhou Julong High-Tech Food Industry Co., Ltd(China), Stevioside (0.07%), and Rebaudioside A (0.028%) were prepared.

The organoleptic test was carried out with 30 previously trained panelmembers. It was observed that Rebaudioside A has the highest sweetnesslevel (5.96), followed by Stevioside with a mean score of 4.62, andcommercial Stevia extract had the lowest mean score of 2.96.Rebaudioside A had the lowest score for bitterness (1.76), andcommercial Stevia extract was the most bitter compared to the othersamples. For overall acceptability, Rebaudioside A had the highest scoreof 4.05 followed by Stevioside (3.81) and raw extract (3.16) (FIG. 1).

The taste profile of Stevioside with 99.3% of purity was more preferableas compared with 90.2 and 95.4% (FIG. 2). The similar feature wasobtained for Rebaudioside A with various grades of purity (FIG. 3).

The highly purified sweeteners can be favorably used for seasoningvarious food products (for instance, soy sauce, soy sauce powder, soypaste, soy paste powder, dressings, mayonnaise, vinegar, powderedvinegar, bakery products and confectioneries, frozen-desserts, meatproducts, fish-meat products, potato salad, bottled and canned foods,fruit and vegetables) in intact or mixed forms with other sweeteners,such as corn syrup, glucose, maltose, sucrose, lactose, aspartame,saccharin, sugar alcohols, organic and amino acids, flavors and/orcoloring agents.

The products are favorably usable as a low-cariogenic and low-caloriesweetener because it is less fermentable by oral dental-caries causativemicroorganisms. Exemplary applications include low-cariogenic foodproducts such as confectioneries including chewing gum, chocolate,biscuits, cookies, toffee and candy. Additionally applications includesoft drinks such as coffee, cocoa, juice, carbonated drinks, sour milkbeverage, yogurt drinks and alcoholic drinks, such as brandy, whisky,vodka and wine. In addition to the above-described uses, the sweetenersare usable for sweetening drugs and cosmetics.

The following examples illustrate preferred embodiments of theinvention.

Example 1 Extraction of Sweet Steviol Glycosides

The leaves of Stevia rebaudiana are dried at 55° C. for three hours in avacuum oven and powdered (30 mesh). One kg of the obtained material wasmixed with 10 liters of water (pH 6.5) and heated to 55° C. with slowagitation for 10 hours. The plant material was separated from thesolution by filtration and the pH of the filtrate was adjusted to 10with about 24 grams of calcium hydroxide and heated to 50° C. for 0.5hours. The obtained mixture was cooled to ambient temperature and the pHwas adjusted to about 7.0 by about 53 grams of FeCl₃. After mixing for15 minutes the precipitate was removed by filtration.

The slightly yellow filtrate was passed through the Celite, deionized,and decolorized by conventional manner on Amberlite FPC23H, AmberliteFPA51, and Amberlite FPA98Cl commercialized by ROHM & HAAS Co., Germany.The solution was concentrated and spray dried. The yield was 122 gramsof powder with a content of sweet glycosides of about 91%. The sweetsteviol glycoside mixture contains 3.4% Dulcoside, 64.6% Stevioside,6.7% Rebaudioside C and 25.3% Rebaudioside A. In another embodiment, asweet steviol glycoside mixture with a purity of at least about 95% wasobtained. In a further embodiment, a sweet steviol glycoside mixturewith a purity of at least about 98% was obtained.

Example 2 Preparation of Stevioside

100 grams (on the base of dry material) of the sweet steviol glycosidepowder obtained by the process of EXAMPLE 1 was mixed with 0.5 liters ofmethanol and maintained at 25° C. for 45 minutes with slow agitation.The precipitate Stevioside was filtered and dried. 61.2 grams ofStevioside with 90.6% purity was obtained.

For the further purification the powder was mixed with two parts of 90%of ethanol, and maintained at 10-12° C. for about 30 minutes with slowagitation. The precipitate was separated by filtration and dried undervacuum. The product weighed 58.8 grams and contained 99.3% Stevioside.In another embodiment, Stevioside with a purity of at least about 98%was obtained.

Example 3 Preparation of Rebaudioside A

The remaining solutions after separation of Stevioside (EXAMPLE 2) werecombined, and methanol was removed by evaporation. The syrup was dilutedwith water and passed through polysulfone based ultrafiltrationmembranes (with a filtering discrimination of 2.5 kDa) (LiumarTechnologies, Ottawa, Canada) with diafiltration. The filtrate wasconcentrated and spray dried. 40.8 grams of powder with content ofRebaudioside A of around 60% were obtained. The powder was mixed withfive volumes (w/v) of 96.2% ethanol and maintained at 50° C. for 30minutes with slow agitation. The precipitate was filtered and dried.Rebaudioside A with 89.8% purity was obtained. The powder was mixed withtwo volumes of 92% of ethanol and maintained at 12° C. for 60 minuteswith slow agitation. The crystals were filtered and dried. 23.6 grams ofRebaudioside A of 98.9% purity was obtained. In another embodiment,Rebaudioside A with a purity of at least about 98% was obtained.

Example 4 Low-Calorie Orange Juice Drink

Orange concentrate (35%), citric acid (0.38%), ascorbic acid (0.05%),sodium benzoate (0.02%), orange red color (0.01%), orange flavor(0.20%), and sweetener (0.06%) containing 90.2, 95.4 or 99.3% ofStevioside, or 80, 90, or 98.9% of Rebaudioside A were blended anddissolved completely in the water (up to 100%) and pasteurized. Thesensory evaluation of the samples is summarized in the TABLE 1. The datashows that best results were obtained for highly purified Rebaudioside Aand Stevioside.

TABLE 1 Comments Sample Flavor Aftertaste Mouth feel Stevioside - Sweetand balanced flavor Slight bitterness in Acceptable 90.2% aftertasteStevioside - Sweet and balanced flavor Slight bitterness in Acceptable95.4% aftertaste Stevioside - Sweet, pleasant, balanced flavor Clean, nobitterness Quite full 99.3% Rebaudioside Sweet, rounded and balancedAlmost no any Acceptable A -80.0% Flavor bitterness Rebaudioside Sweet,rounded and balanced Almost no any Full A -90.0% Flavor bitternessRebaudioside High quality of sweetness, Clean, no unpleasant Quite fullA -98.9% pleasant, taste similar to aftertaste sucrose, balanced flavor

By the same way can be prepared juices from other fruits, such as apple,lemon, apricot, cherry, pineapple, etc.

Example 5 Low-Calorie Carbonated Lemon-Flavored Beverage

The formula for the beverage was as below:

Ingredients Quantity, kg Sugar 30.0 Sweetener 0.4 Citric acid 2.5 Greentea extract 25.0 Salt 0.3 Lemon tincture 10.0 L Juniper tincture 8.0 LSodium benzoate 0.17 Carbonated water up to 1000 L

Sensory and physicochemical characteristics of the drink are presentedin the TABLE 2.

The drinks with highly purified Rebaudioside A and Stevioside weresuperior with an excellent flavor and taste.

TABLE 2 Characteristics Stevioside - Rebaudioside Rebaudioside ItemStevioside - 90.2% 99.3% A - 90.0% A - 98.9% Appearance Transparentliquid, Transparent Transparent Transparent free of sediment liquid,free of liquid, free of liquid, free of and strange sediment andsediment and sediment and impurities. A light strange strange strangeopalescence, caused impurities. A impurities. A impurities. A byfeatures of used light light light raw materials is opalescence,opalescence, opalescence, possible. caused by caused by caused byfeatures of features of features of used raw used raw used raw materialsis materials is materials is possible. possible. possible. Color Fromlight yellow up to From light From light From light Yellow yellow up toyellow up to yellow up to yellow yellow yellow Taste Sour-sweet, someSour-sweet, Sour-sweet, Sour-sweet, bitterness in expression of almostno any expression of aftertaste sweetness is bitterness, sweetness israpid. The expression of rapid. taste is sweetness is satisfactory,rapid.

Example 6 Low-Calorie Carbonated Drink

The formula for the beverage was as below:

Ingredients Quantity, % Cola flavor 0.340 Phosphoric acid (85%) 0.100Sodium citrate 0.310 Sodium benzoate 0.018 Citric acid 0.018 Sweetener0.030 Carbonated water to 100

The beverages prepared with different sweeteners were given to 10 judgesfor comparison.

TABLE 3 shows the results.

TABLE 3 Number of panelists Comparison Stevioside - Stevioside -Rebaudioside A Rebaudioside A Point 90.2% 99.3% 90.0% 98.9% Bitter taste6 2 3 0 Astringent taste 6 2 3 0 Aftertaste 6 2 3 0 Quality of Sweet,Clean (7 of the Sweet, some Clean (10 of the sweet taste bitterness in10 judges) bitterness in 10 judges) aftertaste (6 of aftertaste (5 ofthe 10 judges) the 10 judges) Overall Satisfactory (5 Satisfactory (8Satisfactory (8 Satisfactory (10 evaluation of the 10 of the 10 of the10 of the 10 judges) judges) judges) judges)

The above results show that the beverages prepared using highly purifiedStevioside and Rebaudioside A possessing good organolepticcharacteristics.

Example 7 Chocolate

A composition containing 30 kg of cacao liquor, 11.5 kg of cacao butter,14 kg of milk powder, 44 kg of sorbitol, 0.1 kg of salt, and 0.1 kg ofsweetener prepared according to the EXAMPLES 2 or 3, was kneadedsufficiently, and the mixture was then placed in a refiner to reduce itsparticle size for 24 hours. Thereafter, the content was transferred intoa conche, 300 grams of lecithin was added, and the composition waskneaded at 50° C. for 48 hours. Then, the content was placed in ashaping apparatus, and solidified.

The products are low-cariogenic and low-calorie chocolate with excellenttexture. Also, the organoleptic test carried out with 20 panelistsrevealed no lingering after-taste. The most desirable ones were theproducts with Rebaudioside-98.9% (19 members) and Stevioside 99.3% (16members).

Example 8 Ice-Cream

1.50 kg of whole milk were heated to 45° C., and 300 grams of milkcream, 100 grams of tagatose, 90 grams of sorbitol, 6 grams ofcarrageenan as a stabilizer, 3 grams of polysorbate-80 as an emulsifier,and 1.0 gram of sweetener prepared according to the EXAMPLES 2 or 3,were added into the milk and was stirred until the ingredientscompletely dissolved. The mixture then was pasteurized at a temperatureof 80° C. for 25 seconds. The homogenization of the obtained mixture wascarried out at a pressure of 800 bars and the samples were kept at atemperature of 4° C. for 24 hours to complete the aging process. Vanillaflavor (1.0% of the mixture weight) and coloring (0.025% of the mixtureweight) are added into the mixture after aging. The mixture was thentransferred to ice cream maker to produce ice cream automatically.Samples of ice creams produced were transferred to seal containers andwere kept in the freezer at a temperature of −18° C.

The application of sweeteners does not affect the physicochemicalproperties of ice cream, as well as the overall attributes of color,smoothness, surface texture, air cell, vanilla aroma intensity, vanillataste, chalkiness, iciness and melting rate. Organoleptic test carriedout with 20 panelists. The most desirable ones were the products with98.9% Rebaudioside A (18 members) and 99.3% Stevioside (14 members).

Example 9 Yogurt

In 5 kg of defatted milk, 4.0 grams of sweetener, prepared according toEXAMPLES 2 and 3, were dissolved. After pasteurizing at 82° C. for 20minutes, the milk was cooled to 40° C. A starter in amount of 150 gramswas added and the mixture was incubated at 37° C. for 6 hours. Then, thefermented mass was maintained at 10-15° C. for 12 hours.

The product is a low-calorie and low-cariogenic yoghurt without foreigntaste and odor.

Example 10 Table Top Tablet

A mixture, consisting of 58.5% lactose, 10% calcium silicate, 5%cross-carmellose, 5% L-leucine, 1% aerosol 200, 0.5% magnesium stearate,and 20% of a sweetener, obtained according to the EXAMPLE 2 or 3, waskneaded sufficiently. Then the mixture was shaped with the use of atabletting machine, equipped with punchers of 6.2 mm diameter, intotablets of 70 mg each, 3.0 mm thick, and 10±1 kg hardness

The tablets can be easily administrated due to their appropriatesweetness. However, the formulations using low grade of Stevioside andRebaudioside A were somewhat sticky with solubility about 3-4 minutes inwater at 25° C. The tablets, prepared with highly purified RebaudiosideA show the best characteristics with the solubility around 20-30seconds.

Example 11 Tooth paste

A tooth paste was prepared by kneading a composition comprising ofcalcium phosphate, 45.0%; carboxymethylcellulose, 1.5%; carrageenan,0.5%; glycerol, 18.0%; polyoxyethylene sorbitan mono-ester, 2.0%;beta-cyclodextrin, 1.5%; sodium laurylsarcosinate, 0.2%; flavoring,1.0%; preservative, 0.1%; Rebaudioside A or Stevioside, obtainedaccording to the EXAMPLE 2 or 3, 0.2%; and water to 100%, by usual way.The product possesses good foaming and cleaning abilities withappropriate sweetness.

Example 12 Soy sauce

0.8 g of Rebaudioside AlStevioside mixture (1:1, w/w) obtained accordingto the invention was added to 1000 mL of soy sauce and mixedhomogenously. The products had an excellent taste and texture.

Example 13 Bread

1 kg of wheat flour, 37.38 grams of fructooligosaccharide syrup, 80grams of margarine, 20 grams of salt, 20 grams of yeasts, and 0.25 gramsof high purity Rebaudioside A or Stevioside, obtained according to theEXAMPLE 2 or 3 were placed into the blender and mixed well. 600 ml ofwater was poured into the mixture and kneaded sufficiently. At thecompletion of the kneading process, the dough was shaped and raised for30 to 45 minutes. The ready dough was placed in oven and baked for 45minutes. Bread samples had creamy white color, and smooth texture.

Example 14 Diet Cookies

Flour (50.0%), margarine (30.0%), fructose (10.0%), maltitol (8.0%),whole milk (1.0%), salt (0.2%), baking powder (0.15%), vanillin (0.1%),Rebaudioside A or Stevioside (0.55%), obtained according to thisinvention were kneaded well in dough-mixing machine. After molding ofthe dough the cookies were baked at 200° C. for 15 minutes.

The product is a low-calorie diet cookie with excellent taste andappropriate sweetness.

Example 15 Cake

123 g of hen eggs, 45 g of sugar, 345 g of sorbitol liquid, 2.0 g ofsucrose fatty acid ester, 0.35 g of Rebaudioside A or Stevioside wasmixed with 100 g of wheat flour and 200 g of water in order to prepare acake according to a conventional method. The product had an excellenttaste with an optimal sweet flavor.

The following examples describe a process to make a sucrose-basedsweetener with stevia-based sweeteners having improved taste, mouthfeeland flavor properties. Because sucrose and stevia-based sweeteners havevery different melting characteristics and solubility, conventionalco-crystallization techniques would not result in a suitable product.For example, while sugar is about 66% soluble in water, Rebaudioside Ais only about 1% soluble, and Stevia 95 (containing steviol glycosidesat 95% purity) is only about 34% soluble in water. Sugar has a meltingpoint of about 186° C., while Rebaudioside A has a melting point ofabout 240° C., and Stevia 95 is mostly amorphous. Because of these andother disparities, conventional processes would not lead to a suitableproduct.

Instead, the following process was developed to overcome thedifficulties associated with working with sweetening components havingsuch different properties. The resulting low-calorie sweetener can beused in any food, beverage or consumer healthcare product. Otheringredients can be incorporated into the low-calorie sweetener usingthis process, including but not limited to sugar, salt, vitamins,minerals, supplements, homeopathic agents, preservatives, citric acid,juniper tincture, ascorbic acid, sodium benzoate, colorings, flavorings,and a combination thereof.

Although the processes described in the following Examples makereferences to the sweeteners made in Examples 1-3, it is to beunderstood that these processes can be used with any stevia-basedsweeteners, including but not limited to Rebaudioside A, Stevioside, andpurified sweet steviol glycoside mixtures, made by using any process,including the enzymatic transglycosylation processes described inco-pending U.S. patent application Ser. Nos. 11/246,066, 12/684,129 and12/684,130, the entire contents of which are incorporated by referenceherein. The processes described in these co-pending applications involvean enzymatic transglycosylation process using CGTases (cyclodextringlycosyltransferase) produced by cultures of Bacillus stearothermophilusto produce stevia-based sweeteners, including, but not limited to,Steviosides, Rebaudioside A and purified sweet steviol glycosidemixtures.

Example 16 Sugar (Sucrose) Based Sweetener with Stevioside

1488 g of granulated sugar with moisture content adjusted to 0.80% wasdistributed to form a layer with thickness of 30 mm on a vibrating tray.12.05 g stevioside obtained according to EXAMPLE 2, was dissolved in18.07 g of solvent mixture containing 4 volumes of water per 1 volume ofethyl alcohol to make stevioside 40% (w/w) solution. The solution washeated up to 40° C. for prevention of crystal formation and wasdispersed on the granulated sugar by means of an air-powered pneumaticmethod with pressure of compressed air at 0.1 MPa over a period of 100seconds while maintaining the intensity of vibration at 1200 vibrationsper minute (vpm). The granulated sugar was dried over a period of 10minutes by means of a convective method in a drum-type drying apparatusset at a temperature of 65° C. until its moisture content was 0.09%.

The sweetener produced has a homogenous and intact structure of crystalsugar and has a taste profile identical to sucrose, with sweetness powerabout or at least about 3 times higher than sugar.

Example 17 Sugar (sucrose) based sweetener with Rebaudioside A

1490 g of granulated sugar with moisture content adjusted to 0.90% wasdistributed to form a layer with thickness of 30 mm on a vibrating tray.10.03 g rebaudioside A obtained according to EXAMPLE 3, was dissolved in15.04 g of solvent mixture containing 4 volumes of water per 1 volume ofethyl alcohol to make rebaudioside A 40% (w/w) solution. The solutionwas heated up to 40° C. for prevention of crystal formation and wasdispersed on the granulated sugar by means of an air-powered pneumaticmethod with pressure of compressed air at 0.1 MPa over a period of 100seconds while maintaining the intensity of vibration at 1200 vpm. Thegranulated sugar was dried over a period of 10 minutes by means of aconvective method in a drum-type drying apparatus set at a temperatureof 65° C. until its moisture content was 0.08%.

The sweetener produced has a homogenous and intact structure ofcrystalline sugar and has a taste profile identical to sucrose, withsweetness power about or at least about 3 times higher than sugar.

Example 18

Sugar (Sucrose) based sweetener with Reb A and Steviol glycosidemixtures

A mixture of 50 kg of sugar with 192 gm of Reb A obtained according toEXAMPLE 3 was thoroughly blended in a ribbon blender and then compactedinto sheets of product using a roll compaction unit (IR 520 rollcompactor or similar unit) applying roll speeds ranging between about 12to 16 rpm under a roll pressure of about 14 to 17 bar, which was thenmilled and classified to get optimum sized granules of the sugar-Reb Ablend.

The sugar-Reb A granules had sweetness profile like sugar with about twotimes the sweetness potency of sugar

A mixture of 65 kg of sugar with 188 gm of Steviol Glycoside obtainedaccording to EXAMPLE 1 was thoroughly blended in a ribbon blender andthen compacted into sheets of product using a roll compaction unit (IR520 roll compactor or similar unit) applying roll speeds ranging betweenabout 12 to 16 rpm under a roll pressure of about 14 to 17 bar, whichwas then milled and classified to get optimum sized granules of thesugar-Steviol Glycoside blend.

The sugar-Steviol Glycoside granules had a sweetness profile like sugarwith at least 50% higher sweetness than sugar alone.

Example 19

Sugar based sweetener with Reb A.

A sugar solution of 65 kg of refined sugar in 35 kg of warm water wasmade and mixed thoroughly with 138 gm of Reb A obtained according toEXAMPLE 3. The solution mixture was passed through multiple-effectevaporators, and then to the vacuum pans to reach the desired level ofsuper-saturation, when the sugar liquor is “seeded” with invert sugar toinitiate formation of sugar crystals. The sugar liquor with developingcrystals is then discharged in a mixer to complete the crystallizationand then to a centrifugal for separation of crystals from mother liquor.The separated crystals are then dried in a fluidized bed dryer/cooler.

The co-crystallized sugar-Reb A had sweetness profile like sugar with atleast 50% higher sweetness potency than sugar alone.

Example 20

Sugar (sucrose) based sweetener with a purified sweet steviol glycosidemixture

1488 g of granulated sugar with moisture content adjusted to 0.80% wasdistributed to form a layer with thickness of 30 mm on a vibrating tray.11.58 g of the purified sweet steviol glycoside mixture obtainedaccording to EXAMPLE 1 was dissolved in 17.37 g of solvent mixturecontaining 4 volumes of water per 1 volume of ethyl alcohol to make 40%(w/w) solution. The solution was heated up to 40° C. for prevention ofcrystal formation and was dispersed on the granulated sugar by means ofan air-powered pneumatic method with pressure of compressed air at 0.1MPa over a period of 100 seconds while maintaining the intensity ofvibration at 1200 vpm. The granulated sugar was dried over a period of10 minutes by means of a convective method in a drum-type dryingapparatus set at a temperature of 65° C. until its moisture content was0.05%.

The sweetener produced has a homogenous and intact structure ofcrystalline sugar and has taste profile identical to sucrose withsweetness power 3 times higher than sugar.

In one embodiment, the dry weight ratio of sucrose to the sweet steviolglycoside mixture, stevioside or rebaudioside A is in the range of about1:50 to about 1:300 (w/w). Examples of other ratios of sucrose to eithersteviol glycosides (“Stevia95” which contains 95% total steviolglycosides), stevioside or rebaudioside A (Reb A) suitable for thepresent invention are summarized in Table 4. As used in the Table,“Calorie Reduction” refers to the reduced calories of the sweetener ascompared to 100 kg of pure sugar. Each of the following blends providesthe equivalent sweetness of 100 kg of sugar.

TABLE 4 “Stevia Cal- 95” Steviol orie RebA Stevioside glycoside mixtureRe- Sugar Sugar to Sugar to duc- Sugar Weight to RebA Weight SteviosideWeight Stevia tion (kg) (kg) ratio (kg) ratio (kg) 95 ratio 25% 75 0.096780 0.132 570 0.119 630 35% 65 0.135 483 0.188 345 0.167 390 50% 500.192 260 0.278 180 0.238 210 75% 25 0.357 70 0.480 52 0.429 58

It is to be understood that the foregoing descriptions and specificembodiments shown herein are merely illustrative of the best mode of theinvention and the principles thereat and that modifications andadditions may be easily made by those skilled in the art withoutdeparting for the spirit and scope of the invention, which is thereforeunderstood to be limited only by the scope of the appended claims.

1) A low-calorie sweetener composition, comprising: a low intensitysweetener; and a high intensity sweetener which is applied onto the lowintensity sweetener so as to form the low-calorie sweetener composition.2) The low-calorie sweetener composition of claim 1, wherein the lowintensity sweetener is selected from the group consisting of sucrose,glucose, maltose, fructose, lactose, tagatose, palatinose, sugaralcohols, and combinations thereof. 3) low-calorie sweetener compositionof claim 1, wherein the high intensity sweetener is selected from thegroup consisting of: steviol glycosides including a purified sweetsteviol glycoside mixture, stevioside, rebaudioside A, rebaudioside B,rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F,dulcoside A, dulcoside B, rubusoside, and stevia; siamenoside; mogrosideIV; mogroside V; Luo Han Guo sweetener; monatin and its salts (monatinSS, RR, RS, SR); glycyrrhizic acid and its salts; curculin; thaumatin;monellin; mabinlin; brazzein; hernandulcin; phyllodulcin; glycyphyllin;phloridzin; trilobtain; baiyunoside; osladin; polypodoside A;pterocaryoside A; pterocaryoside B; mukurozioside; phlomisoside I;periandrin I; abrusoside A; cyclocarioside I; and combinations thereof.4) The low-calorie sweetener composition of claim 1, wherein the highintensity sweetener comprises one or more of: a purified sweet steviolglycoside mixture; a stevioside; rebaudioside A; or a combinationthereof. 5-13. (canceled) 14) The low-calorie sweetener composition ofclaim 1, further comprising one or more additional components selectedfrom the group consisting of sugar, salt, citric acid, juniper tincture,ascorbic acid, sodium benzoate, coloring, and flavoring. 15) Thelow-calorie sweetener composition of claim 1, wherein the low intensitysweetener to high intensity sweetener dry weight ratio is in the rangeof about 50:1 to about 10000:1 (w/w). 16) A process for producing thelow-calorie sweetener composition of claim 4, comprising: a) adjusting amoisture content of sugar granules to a level of about 0.5% to about1.5%; b) distributing the sugar granules on a vibrating surface to forma layer with thickness of about 10 to about 100 mm on the vibratingsurface; c) dissolving the high intensity sweetener in an aqueousalcohol mixture with an alcohol content of about 0.1% to about 99.9%(v/v) to obtain a solution with about 10% to about 70% (w/w) solids tomake a high intensity sweetener solution; d) dispersing the highintensity sweetener solution onto the sugar granules while maintainingan intensity of vibration at about 1 to about 2000 vpm; e) drying thegranules to form the low-calorie sweetener composition. 17-25.(canceled) 26) The low-calorie sweetener of claim 1, wherein the lowintensity sweetener to high intensity sweetener dry weight ratio is inthe range of about 50:1 to about 300:1 (w/w). 27-30. (canceled) 31) Aprocess for making the low-calorie sweetener of claim 4, comprising thesteps of: a) making a sweetener blend by combining the low intensitysweetener with the high intensity sweetener in a ribbon blender; b)compacting the sweetener blend into a sweetener sheet using a rollcompaction unit; and c) milling the sweetener sheet into granules tomake the low-calorie sweetener. 32) A process for making the low-caloriesweetener of claim 4, comprising the steps of: a) making a sweetenersolution by combining the low intensity sweetener, the high intensitysweetener and water; b) passing the sweetener solution throughmultiple-effect evaporators to make an evaporated sweetener solution; c)applying a vacuum to the evaporated sweetener solution to make asuper-saturated solution; d) seeding the super-saturated solution withan additional amount of the low intensity sweetener to initiate crystalformation; e) allowing crystal formation to continue to completecrystallization in the solution; and f) separating the crystals from thesolution to result in the low-calorie sweetener.